Embryonic stein cells (ESC) are derived from the inner cell mass of pre-implantation embryos, and have been recognized as the most pluripotent stem cell population. These cells are capable of unlimited, undifferentiated proliferation in vitro, and still maintain the capacity for differentiation into a wide variety of somatic and extra-embryonic tissues. Detection and identification of the cell type of interest is a critical step in the stem cell work flow. For example, during the process of somatic reprogramming, several colonies are obtained out of which only a few are truly pluripotent. Additionally stem cell populations are dynamic culture systems with the propensity for differentiation. For example, pluripotent stem cells such as ESC and induced pluripotent stem cells (iPSC) are characterized periodically to qualify their undifferentiated, pluripotent state. Current methods for detection/identification and characterization of pluripotent stem cells are primarily immunochemical staining methods, using antibodies against unique surface markers, which may be terminal and expensive.
Alternate methods include detection using alkaline phosphatase activity. Alkaline phosphatase is an enzyme in the blood, intestines, liver, and bone cells, and exists as membrane-bound isoforms of glycoproteins sharing a common protein structure but differing in carbohydrate content. These enzymes are most active at alkaline pH. Undifferentiated human pluripotent stem cells have been shown to express a very high level of alkaline phosphatase.
However, known substrates and methods for staining stem cells, including alkaline phosphatase staining methods, are generally terminal staining methods (i.e. toxic to the cells), and therefore are limiting as the stained colonies cannot be propagated further. Accordingly, there is a need in the industry for novel substrates and methods for staining stem cells using a solution that is both cell-permeable and nontoxic, and that specifically stains pluripotent stem cells without altering survival or stem cell characteristics. The invention described herein may, in various embodiments, solve some or all of these needs. The basic principle reported in this application can be extended to other cell types based on differential expression of a functional protein via delivery of the appropriate substrate that preserves the cells in their live condition.